Over the past five years this laboratory has concentrated on studies of the flavin-containing monooxygenase (FMO), a drug-metabolizing enzyme. We have defined the FMO gene family in the rabbit and have derived the sequences of the five gene products from cloned cDNAs. The identities among the primary structures are all near 55% and each gene product represents a distinct gene subfamily. Expression of all five of the FMOs exhibits some degree of tissue selectivity that varies from species to species. For example, the form of the enzyme formerly referred to as the "liver" FMO predominates in the kidney of rodents. We have recently expressed the rabbit pulmonary FMO (FMO 1B1) in E. coli and found that this system can be used to examine structure/function relationships. In an attempt to understand the molecular bases for the differences between FMO 1A1 and FMO 1B1, residues N469, S470, Q480, K492 and Q493, uniquely conserved in FMO 1B1 were substituted with the residues present in FMO 1A1 (T, P, K, W and D). All of the mutants except the tandem mutant at 492-493 were stimulated by NaCholate and imipramine in the same manner as observed for the wild type. A rabbit liver genomic DNA library was screened with a mixture of cDNA probes from rabbit FMOs 1A1, 1B1, 1C1 and 1D1. Twenty positive clones purified to homogeneity were identified by hybridization with individual probes as 1A1 (7), 1B1 (1), 1C1 (5), and 1D1 (7). An FMO 1A1 clone that also hybridized with middle and 3' probes was chosen for further characterization. The 17 kB gene fragment was digested with Hind III, and the resulting fragments (1.0 to 5.9 kB) subcloned into Bluescript phagemid and sequenced with T3 and T7 primers, and primers complementary to FMO 1A1. With approximately 90% of the coding region sequenced, six introns with characteristic exon/intron junctions have been identified. The largest exon (0.7 kB) is located at the 3'-end and contains the stop codon as well as a putative polyadenylation signal; the smallest exon is only 72 bases in length. Attempts are now being made to characterize the 5'-flanking region of the gene.